Pathogen for bacterial poultry disease

ABSTRACT

The invention belongs to the field of animal health and in particular the causative agent of a new bacterial poultry disease,  Gallibacterium . The invention provides said  Gallibacterium  bacteria, vaccine comprising inactivated  Gallibacterium , and a method of immunizing to prevent disease in poultry.

RELATED APPLICATIONS

This application is a continuation application of currently pending Ser.No. 12/548,210 filed Aug. 26, 2009, which is a continuation ofapplication Ser. No. 12/392,594 filed Feb. 25, 2009, now U.S. Pat. No.7,794,735 issued Sep. 14, 2010, which is a continuation of applicationSer. No. 10/279,732 filed Oct. 24, 2002, now U.S. Pat. No. 7,521,060issued Apr. 21, 2009, which is a nonprovisional of application Ser. No.60/348,253 filed Nov. 7, 2001, which is expired, which claims priorityto German application S/N 101 52 370, filed Oct. 26, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention belongs to the field of animal health and in particularthe causative agents of a new bacterial poultry disease, Gallibacterium.The invention provides said Gallibacterium bacteria, a vaccinecomprising inactivated Gallibacterium, and a method of immunizingchicken to prevent said disease in chicken.

2. Description of the Related Art

During the last decade, intensive poultry farming methods to increaseproductivity, has resulted in an increase of disease manifestationthroughout all major poultry producing countries. This has caused anincreasing need for new and better vaccines and vaccination programs tocontrol these diseases. Nowadays, most animals are immunized against anumber of diseases of viral and bacterial origin.

Examples of viral diseases in poultry are Newcastle Disease, InfectiousBronchitis, Avian Pneumovirus, Fowlpox, Infectious Bursal Disease etc.

Examples of bacterial diseases are Avian Coryza caused by Haemophilusparagallinarum (upper respiratory tract), Bordetella avium (upperrespiratory tract), Ornithobacterium rhinotracheale (lower respiratorytract), Salmonella infections (digestive tract), Pasteurella multocida,which is the causative agent of fowl cholera (septicemic), and E. coliinfections.

Therefore, the technical problem underlying this invention was toidentify a new bacterial poultry disease, to provide the causative agentof said disease and to provide a vaccine to prevent said disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. The application contains at leastone drawing executed in color. Copies of this patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

A) From Field Outbreaks

FIG. 1) Broilers: Nasal discharge and swollen areas around the eye.

FIG. 2) Broilers: Haemorrhage in heart and coronary fat.

FIG. 3) Broilers: Conjuctivitis and inflammation around the eye.

FIG. 4) Layers: Nasal discharge and displaced comb with cyanosis.

FIG. 5) Layers: Inflammation and haemorrhage around the eye.

FIG. 6) Layers: Haemorrhage in the dermal tissue behind entrance toauditory orifice.

FIG. 7) Layers: Inflammation of kidneys.

FIG. 8) Layers: Haemorrhages in oviduct.

FIG. 9) Layers: Deformed ovarian follicles.

FIG. 10) Layers: Haemorrhage in the junction between proventriculus andgizzard.

FIG. 11) Layers: Congestion and haemorrhage in oviduct.

B) Experimental Infection

FIG. 12) Layers: Inflammation and haemorrhage in kidney.

FIG. 13) SPF: Prostration.

FIG. 14) Layers: Haemorrhage in joint and muscle.

FIG. 15) Layers: Nasal discharge and pale comb.

FIG. 16) Layers: Haemorrhage in muscle.

FIG. 17) SPF: Haemorrhage in heart and coronary fat.

FIG. 18) Layers: Healthy bird on the left and sick bird on the rightwith ruffled feathers.

FIG. 19) Layers: Greenish diarrhea.

FIG. 20) SPF: Haemorrhage in muscle.

FIG. 21) SPF: Prostration (locomotive problems) and greenish diarrhea.

FIG. 22) Layers: Enlarged liver with haemorrhage.

DISCLOSURE OF THE INVENTION Definitions of Terms Used in the Description

Before the embodiments of the present invention it must be noted that asused herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a Gallibacterium” includes aplurality of such Gallibacterium bacteria, reference to the “cell” is areference to one or more cells and equivalents thereof known to thoseskilled in the art, and so forth. It is irrelevant whether a word iscapitalized or not, therefore both “Arabinose” and “arabinose” have thesame meaning, unless indicated otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods, devices, andmaterials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the cell lines, vectors, and methodologies as reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Surprisingly, a new bacterial poultry disease has been observed by thepresent inventors, which occurs primarily in layers and less frequent inbroilers. The disease was seen in chicken that had been vaccinatedagainst the bacterium Haemophilus paragallinarum (causative agent ofavian Coryza), and Pasteurella multocida (causative agent of fowlcholera). The symptoms of this new disease differ from the specificsymptoms of Coryza. Given the fact that the newly discovered diseaseclearly shows the clinical signs of a upper respiratory tract infectionas described below, H. paragallinarum could be ruled out as thecausative agent.

The present invention relates in a first embodiment to Gram-negative,facultative anaerobic, pleomorphic rod-shaped bacteria causing a noveldisease of the upper respiratory and of the reproductive tract ofpoultry, wherein said bacteria are selected from Gallibacterium.

Said bacteria according to the invention may be isolated from infectedtrachea, palatine cleft, ovary, liver, heart, kidney and gonads(broilers). They can be identified as Gallibacterium according to theinvention based on the tests listed below:

Beta haemolysis + Gram stain − Oxidase + Catalase + Urease NitrateIndole

The bacterial isolates are preferably purified and biotyped according tothe method initially proposed by Christensen et al. (Christensen, H.,Bisgaard, M., Bojesen, A. M., Mutters, R., and Olsen, J. E., Geneticrelationships among avian isolates classified as Pasteurellahaemolytica, ‘Actinobacillus salpingitidis’ or Pasteurella anatis withproposal of Gallicaterium anatis gen. nov., comb. nov. and descriptionof additional genomospecies within Gallibacterium gen. nov., Int. J.Syst. Evol. Microbiol., 2003, 53, 275-287). Said methods may be appliedby the artisan to find out whether bacteria are within the scope of thepresent invention.

Previously, the bacterial isolates were purified and biotyped accordingto the method described by Jaworski et al. (Jaworski M. D., D. L.Hunter, A. C. S. Ward. Biovariants of isolates of Pasteurella fromdomestic and wild ruminants. J. Vet. Invest. 1988, 10: 49-55.). Thismethod is also exemplified in the examples Important method to classifybacteria are DNA-DNA hybridization, REA (restriction enzyme analysis seee.g. J. Clinical Microbiol, 1993, 31: 831-835) and ribotyping. Achallenge model to validate Koch's postulates is also exemplified in theexamples.

Thus, an important embodiment of the present invention areGallibacterium, wherein said Gallibacterium arebeta(β)-haemolysis-positive, Gram-negative, oxidase-positive,catalase-positive, urease-negative, nitrate-positive andindole-negative. Preferably, said Gallibacterium according to theinvention are also MacConkey-positive. Even more preferred, saidGallibacterium according to the invention are additionallyGlucose-positive, Sucrose-positive, Mannitol-positive,Arabinose-negative, Celobiose-negative, Xylose-positive,Salicin-negative, Ornithine-negative, Esculin-negative,alpha-Fucosidase-negative, beta-Galactosidase-positive. Most preferredare Gallibacterium according to the invention, wherein saidGallibacterium are also Arabinose-negative and Trehalose-positive.Preferably also, said Gallibacterium according to the invention are alsobeta(β)-Glucosidase-negative or -positive, depending on the biotype.Also most preferred are Gallibacterium according to the invention,wherein said Gallibacterium are furthermore Arabinose-negative andTrehalose-negative. Preferably also, said Gallibacterium according tothe invention are also beta-Glucosidase negative.

These characteristic properties of the bacteria according to theinvention renders the bacteria according to the invention novel overother known bacterial poultry pathogens (Diseases of Poultry, TenthEdition, Edited by B. W. Calnek, Iowa State University Press, Iowa,U.S.A. 1997).

Another preferred embodiment of the present invention are Gallibacteriumaccording to the invention, wherein said poultry is selected from thegroup of chicken, turkey, duck, goose, dove, pigeon and quail.

The invention provides a novel type of Gram-negative, facultativeanaerobic, pleomorphic rod-shaped bacteria, said novel type of bacteriabeing characterized by the bacteria deposited at the American TypeCulture Collection (ATCC), 1081, University Boulevard, Manassas, Va.20110-2209, USA, under the following deposit numbers:

ATCC No. PTA-3667 (internal designation BIV-4985);

ATCC No. PTA-3668 (internal designation BIV-AVICOR);

ATCC No. PTA-3669 (internal designation BIV-07990).

The date of deposit was Aug. 22, 2001.Thus, a most preferred embodiment of the present invention areGallibacterium as deposited at the under accession number ATCC No.PTA-3667. These bacteria are further exemplified in table 3 of example1.

Another most preferred embodiment of the present invention areGallibacterium as deposited at the under accession number ATCC No.PTA-3668. These bacteria are further exemplified in table 2 of example1.

Another most preferred embodiment of the present invention areGallibacterium as deposited at the under accession number ATCC No.PTA-3669. These bacteria are further exemplified in table 1 of example1.

In light of the on-going terminology changes in the art, the strains ofthe present application were subjected to phenotypical characterizationaccording to Christensen et al. (2003), including gram, urase enzymeactivity, motility, cytochrome oxidase activity, and haemolysis.Furthermore, all the strains were hybridized both withGallibacterium-specific probe GAN850, and its inverse and complementaryprobe, and the Gallibacterium-specific probe EUB338 according to Bojesenet al., Detection of Gallibacterium spp. In Chickens by Fluorescent 16SrRNA In Situ Hybridization, J. Clin. Microbiol., Vol. 41, No. 11,November 2003, p. 5167-5172. The results from both phenotypic andgenotypic characterization indicated that all the strains belonged togenus Gallibacterium.

Under the previous method described by Jaworski et al., the results oftests from sections A and B in the examples (Tables 1, 2 and 3)initially indicated that the bacteria BIV-4895; ATCC No. PTA-3667 andBIV-AVICOR; ATCC No. PTA-3668 belonged to the family Pasteurellaceae,genus Pasteurella (Pasteurella trehalosi, which are Trehalose positiveand arabinose negative), while the bacteria BIV-07990; ATCC No. PTA-3669belonged to the family Pasteurellaceae, genus Mannheimia (Mannheimiahaemolytica, which are Trehalose negative and arabinose negative).

The invention also relates to microbiological culture comprisingbacteria according to the invention as disclosed above. The culture maybe made by growing said bacteria at a temperature of between 35° and 37°C. The bacteria may be grown under normal atmospheric oxygen pressure.The bacteria can be grown in a variety of different general-purposebacterial growth promoting media known to the skilled person, e.g.Tryptose Broth (TB), Soy Trypticasein Broth or Brain Heart Infusionbroth or any enriched media. The bacteria may also be grown on sheepblood agar incubated at 37° C. for 24 hours.

Various physical and chemical methods of bacterial inactivation areknown in the art. Examples of physical inactivation are UV-radiation,X-ray radiation, gamma-radiation and heating. Examples of inactivatingchemicals are beta-propiolactone, glutaraldehyde, beta-ethyleneimine andformaldehyde.

Preferably the bacteria according to the invention are inactivated withformaldehyde. Surprisingly, the use of formaldehyde at a finalconcentration of 0.2% is an excellent method to inactivate the bacteriaaccording to the invention.

Thus, in another important aspect, the invention relates to a method forinactivation of a Gallibacterium according to the invention comprisingthe use of formaldehyde at a final concentration of 0.2%.

Said bacteria according to the invention which are inactivated by themethods disclosed supra and by other methods of inactivating thebacteria known to the skilled person are embodied in the presentinvention. Therefore, another important aspect are inactivatedGallibacterium obtainable by a method according to the invention or by amethod known in the art. Preferably, said inactivated Gallibacteriumaccording to the invention are selected from the group of ATCC No.PTA-3667, ATCC No. PTA-3668 and/or ATCC No. PTA-3669.

Therefore, another important aspect are live attenuated Gallibacteriumobtainable by a method known in the art. Preferably, said liveattenuated Gallibacterium according to the invention are selected fromthe group of ATCC No. PTA-3667, ATCC No. PTA-3668 and/or ATCC No.PTA-3669. Said Gallibacterium according to the invention are attenuatedby multiple passages in appropriate culture media or by any other methodknown in the art.

Inactivated as understood herein means, that the Gallibacteriumaccording to the invention are killed without possible replication tocause clinical disease.

Attenuated as understood herein means, that the Gallibacterium accordingto the invention are live bacteria with possible replication but willnot cause clinical disease.

Yet another important aspect are fractions or fragments ofGallibacterium obtainable by a method known in the art. Said fragmentsmay be prepared by detergent solubilization of Gallibacterium accordingto the invention or by any other method known in the art.

Preferably, said fractions or fragments are purified antigens of saidGallibacterium according to the invention. Preferably, saidfractions/fragments are outer membrane proteins of Gallibacteriumaccording to the invention.

A “fragment” according to the invention is any immunogenic subunit of asaid Gallibacterium according to the invention, i.e. any polypeptidesubset.

Thus, the invention relates to fragments containing at least one antigenof Gallibacterium according to the invention. Most preferably, saidfragments are containing at least one antigen of bacteria selected fromthe group of ATCC No. PTA-3667, ATCC No. PTA-3668 and/or ATCC No.PTA-3669. Said fragment may comprise whole bacterial cells of saidstrain(s), bacterial extracts, Outer Membrane Fractions, bacterial exo-and/or endotoxins, and purified proteins. Antigenic polypeptides orfragments thereof may for example be obtained from purified bacterialproteins or by expression of the corresponding genetic material in someprokaryotic or eukaryotic expression system or by organo-chemicalsynthesis. Said methods are known to the skilled person.

The invention further relates to live, and/or live attenuated, and/orinactivated Gallibacterium according to the invention and/or fractionsof said Gallibacterium for use in a vaccine.

The invention further provides a vaccine derived from the newlyidentified bacteria disclosed above. Thus, the invention further relatesto a vaccine composition comprising a live, and/or live attenuated,and/or inactivated Gallibacterium according to the invention and/orfractions of said Gallibacterium.

The term “vaccine” as understood herein is a vaccine for veterinary usecomprising antigenic substances and is administered for the purpose ofinducing a specific and active or passive immunity against a diseaseprovoked by said Gallibacterium. The live or live attenuatedGallibacterium according to the invention confer active immunity thatmay be transferred passively via maternal antibodies against theimmunogens it contains and sometimes also against antigenically relatedorganisms. The inactivated Gallibacterium according to the inventionand/or fractions of said Gallibacterium confer passive immunity.

Additional components to enhance the immune response are constituentscommonly referred to as adjuvants, like e.g. aluminium hydroxide,mineral or other oils or ancillary molecules added to the vaccine orgenerated by the body after the respective induction by such additionalcomponents, like but not restricted to interferons, interleukins orgrowth factors.

In a preferred embodiment, said vaccine comprises inactivated bacteria.

Preferably, a vaccine of the invention refers to a vaccine as definedabove, wherein one immunologically active component is a liveGallibacterium. The term “live vaccine” refers to a vaccine comprising aparticle capable of division/multiplication.

Preferably also, a vaccine according to the invention comprisesattenuated Gallibacterium according to the invention and apharmaceutically acceptable carrier or excipient. Said vaccine may alsobe administered as a combined vaccine comprising two or more strains ofsaid live, and/or live attenuated, and/or inactivated Gallibacteriumaccording to the invention and/or fractions of two or more strains ofsaid Gallibacterium. Most preferably said live, and/or live attenuated,and/or inactivated Gallibacterium according to the invention and/orfractions of said Gallibacterium in the vaccine are selected from thegroup of ATCC No. PTA-3667, ATCC No. PTA-3668 and/or ATCC No. PTA-3669.

Preferably also, a vaccine according to the invention comprisesinactivated Gallibacterium according to the invention and apharmaceutically acceptable carrier or excipient. Said vaccine may alsobe administered as a combined vaccine comprising two or more strains ofsaid inactivated Gallibacterium

Furthermore, fractions of whole cells may also be used as the relevantimmunogen in the vaccine according to the invention. Therefore,preferably a vaccine according to the invention comprises fractions ofGallibacterium according to the invention and a pharmaceuticallyacceptable carrier or excipient. Said vaccine may also be administeredas a combined vaccine comprising two or more strains of said inactivatedGallibacterium. In particular, the invention relates to vaccinescomprising fragments which contain at least one antigen ofGallibacterium according to the invention. Most preferably, theinvention relates to vaccines comprising fragments which contain atleast one antigen of bacteria selected from the group of ATCC No.PTA-3667, ATCC No. PTA-3668 and/or ATCC No. PTA-3669. Said fragment maycomprise whole bacterial cells, bacterial extracts, Outer MembraneFractions, bacterial exo- and/or endotoxins, and purified proteins.Antigenic polypeptides or fragments thereof may for example be obtainedfrom purified bacterial proteins or by expression of the correspondinggenetic material in some prokaryotic or eukaryotic expression system orby organo-chemical synthesis. Said methods are known to the skilledperson.

Preferably, the vaccine according to the invention also comprises anadjuvant. Therefore, the invention further relates to a vaccinecomposition according to the invention, further comprising one or moresuitable adjuvant(s) and/or excipient(s) and/or carrier(s).

Adjuvants as used herein comprise substances that boost the immuneresponse of the injected animal. A number of different adjuvants areknown in the art. Adjuvants as used herein include Freund's Complete andIncomplete Adjuvant, vitamin E, non-ionic block polymers,muramyldipeptides, Quil A, mineral and non-mineral oil, vegetable oil,and Carbopol (a homopolymer). In a preferred embodiment, the vaccineaccording to the invention bacterin comprises a water-in-oil emulsionadjuvant. Said vaccine is also called a bacterin comprising inactivated(killed) bacteria according to the invention and a water-in-oil emulsionadjuvant. Other ways of adjuvating the bacteria known to the skilledperson are also embodied in the present invention.

Also preferably, the vaccine according to the invention may comprise oneor more suitable emulsifiers, e.g. Span or Tween.

Preferably also, said live, and/or live attenuated, and/or inactivatedGallibacterium according to the invention and/or fractions of saidGallibacterium in the vaccine are selected from the group of ATCC No.PTA-3667, ATCC No. PTA-3668 and/or ATCC No. PTA-3669.

Preferably, the vaccine in the present invention comprises at least oneantigen of bacteria selected from the group of ATCC No. PTA-3667, ATCCNo. PTA-3668 and/or ATCC No. PTA-3669. Said vaccine may comprise wholebacterial cells of said strain(s), bacterial extracts, Outer MembraneFractions, bacterial exo- and/or endotoxins, and purified proteins.Antigenic polypeptides or fragments thereof may for example be obtainedfrom purified bacterial proteins or by expression of the correspondinggenetic material in some prokaryotic or eukaryotic expression system orby organo-chemical synthesis. Said methods are known to the skilledperson.

Most preferably, the invention further relates to a vaccine compositionaccording to the invention, further comprising at least one otherantigen from a virus or microorganism pathogenic to poultry. Preferably,said antigen is in the form of live, attenuated or inactivated virusesor microorganisms or fragments thereof. Said fragment may comprise wholebacterial cells or viral particles, bacterial extracts, viral antigens,viral subunits, Outer Membrane Fractions, bacterial exo- and/orendotoxins, and purified proteins. Antigenic polypeptides or fragmentsthereof may for example be obtained from purified bacterial proteins orby expression of the corresponding genetic material in some prokaryoticor eukaryotic expression system or by organo-chemical synthesis. Saidmethods are known to the skilled person.

Most preferably, the invention further relates to a vaccine compositionaccording to the invention, further comprising at least one otherantigen from a virus or microorganism pathogenic to poultry, whereinsaid virus or microorganism is selected from, but not restricted to, thegroup consisting of Infectious Bronchitis Virus, Newcastle DiseaseVirus, Infectious Bursal Disease Virus (disease: Gumboro), ChickenAnaemia agent, Avian Reovirus, Mycoplasma gallisepticum, AvianPneumovirus, Haemophilus paragallinarum (disease: Coryza), ChickenPoxvirus, Avian Encephalomyelitis virus, Pasteurella multocida and E.coli.

A “pharmaceutical composition” essentially consists of one or moreingredients capable of modifying physiological e.g. immunologicalfunctions of the organism it is administered to, or of organisms livingin or on the organism. The term includes, but is not restricted toantibiotics or antiparasitics, as well as other constituents commonlyused to achieve certain other objectives like, but not limited to,processing traits, sterility, stability, feasibility to administer thecomposition via enteral or parenteral routes such as oral, intranasal,intravenous, intramuscular, subcutaneous, intradermal or other suitableroute, tolerance after administration, controlled release properties.Thus, in another important aspect of the invention the invention relatesto a pharmaceutical composition comprising a live, and/or liveattenuated, and/or inactivated Gallibacterium according to the inventionand/or fractions of said Gallibacterium.

The invention relates to a method of treating a Gallibacterium-infectedanimal (e.g. the live bacteria as described above) belonging to thegroup of poultry wherein said live, attenuated, inactivatedGallibacterium and/or fractions and/or fragments thereof according tothe invention as described supra, are administered to the poultry animalin need thereof at a suitable doses as known to the skilled person andthe reduction of symptoms caused by said Gallibacterium infection ismonitored. Said treatment preferably may be repeated.

Yet another important embodiment is a method of immunizing poultryagainst the disease of the respiratory and reproductive tract caused bya Gallibacterium (e.g. the live bacteria as described above) comprisingadministration of an immunologically effective amount of a vaccineaccording to the invention and the reduction of symptoms caused by saidGallibacterium infection is monitored.

Another important embodiment is the use of an inactivated Gallibacteriumaccording to the invention and/or live Gallibacterium according to theinvention and/or live attenuated Gallibacterium according to theinvention and/or fragments or fractions of said Gallibacterium accordingto the invention in the manufacture of a vaccine for the prophylaxis ofGallibacterium infections.

The invention also relates to a method of diagnosis of a disease causedby comprising the steps of obtaining a sample from poultry, wherein saidsample is selected from the group of blood, serum, plasma, tissuescrapings, washings, swabs, tissue, analysing said sample for thepresence of Gallibacterium according to the invention.

In a preferred embodiment the presence of Gallibacterium is determinedby an immune test. An immune test uses monoclonal antibodies orpolyclonal antisera specific to Gallibacterium. The generation ofmonoclonal antibodies is known in the art (Kearney, J. F., Radbruch A.,Liesegang B., Rajewski K. A new mouse myeloma cell line that has lostimmunoglobulin expression but permits construction of antibody-secretinghybrid cell lines. J. Immunol. 1979, 123: 1548-1550., Köhler, G.,Milstein, C. Continous culture of fused cells secreting antibody ofpredefined specifity. Nature 1975, 265: 495-497) Immune tests includethe methods of detection known in the art such as the ELISA test(enzyme-linked immuno-sorbent assay) or the so-called sandwich-ELISAtest, dot blots, immunoblots, radioimmuno tests (radioimmunoassay RIA),diffusion-based Ouchterlony test or rocket immunofluorescent assays) oragglutination tests (rapid plate or micro-plate agglutination tests).Another immune test is the so-called Western blot (also known as Westerntransfer procedure or Western blotting). The purpose of Western blot isto transfer proteins or polypeptides separated by polyacrylamide gelelectrophoresis onto a nitrocellulose filter or other suitable carrierand at the same time retain the relative positions of the proteins orpolypeptides obtained from the gel electrophoresis. The Western blot isthen incubated with an antibody which specifically binds to the proteinor polypeptide under consideration. These methods of detection can beused by the average skilled person to perform the invention describedherein. Literature references in which the skilled person can find theabove-mentioned methods and other detection methods are listed asfollows: An Introduction to Radioimmunoassay and Related Techniques,Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullocket al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla.Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, Practice andTheory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers, Amsterdam, TheNetherlands (1985).

In another, most particular embodiment, the sample as disclosed supra isincubated with antibodies which are specific to Gallibacterium and theantigen/antibody complex thereby formed is determined

In a particularly preferred embodiment of the method according to theinvention, the presence of Gallibacterium in a sample as disclosed suprais determined by molecular biology methods. Molecular biology methods asused herein means detection methods which include, for example,polymerase chain reaction (PCR), reverse transcriptase polymerase chainreaction (RT-PCR) or may be Northern or Southern blots which the skilledperson can find in the standard reference books (e.g. Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. and Bertram, S, andGassen, H. G. Gentechnische Methoden, G. Fischer Verlag, Stuttgart, NewYork, 1991).

The invention also includes a diagnostic test kit according to theinvention which is characterised in that it contains all the necessaryelements for detecting Gallibacterium.

A diagnostic test kit is a collection of all the components for carryingout a method of diagnosis according to the invention. Some examples (notan exhaustive list) of other elements for performing a method accordingto the invention include containers such as 96-well plates or microtitreplates, test tubes, other suitable containers, surfaces and substrates,membranes such as nitrocellulose filter, washing reagents and buffers. Adiagnostic test kit may also contain reagents which may detect boundantibodies, such as for example labelled secondary antibodies,chromophores, enzymes (e.g. conjugated with antibodies) and thesubstrates thereof or other substances which are capable of bindingantibodies.

The invention further relates to a diagnostic test kit according to theinvention which is characterized in that it contains all the necessaryelements for carrying out a PCR or RT-PCR to detectGallibacterium-specific DNA or RNA. Said kit may contain, but is notlimited to in addition to test tubes or 96-well plates or microtitreplates, other suitable containers, surfaces and substrates, membranessuch as nitrocellulose filters, washing reagents and reaction buffers(which may vary in pH and magnesium concentrations), sterile water,mineral oil, BSA (bovine serum albumin), MgCl₂, (NH₄)₂SO₄, DMSO(dimethylsulphoxide), mercaptoethanol, nucleotides (dNTPs), enzymes suchas Taq-polymerase and reverse transcriptase and, as the DNA matrix, theDNA or cDNA specific for Gallibacterium, oligonucleotides specific for aGallibacterium DNA or RNA, control template, DEPC-water, DNAse, RNAseand further compounds known to the skilled artisan. Oligonucleotidesaccording to the invention are short nucleic acid molecules from about15 to about 100 nucleotides long, which bind under stringent conditionsto the nucleic acid sequence which is complementary to a Gallibacteriumprotein. By stringent conditions the skilled person means conditionswhich select for more than 85%, preferably more than 90% homology (cf.Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd)ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. andBertram, S, and Gassen, H. G. Gentechnische Methoden, G. Fischer Verlag,Stuttgart, New York, 1991).

The following examples serve to further illustrate the presentinvention; but the same should not be construed as limiting the scope ofthe invention disclosed herein.

EXAMPLE I Field Disease Outbreaks Associated with GallibacteriumClinical Signs From Field Observations

Layers Broilers Mild upper respiratory/reproductive Severe upperrespiratory/reproductive Age: 22 weeks of age Age: 7 weeks Nasaldischarge Sneezing with rales Swollen areas around the eye Swollen headLow feed consumption Low feed consumption Whitish diarrhea DepressionDecreased egg production Non uniform growth Low mortality Ruffledfeathers Comb displaced Prostration Cyanosis of comb Mortality 8%

Gross Lesions Ovarian atrophy with haemorrhages Haemorrhages in gonadsand regression Ovarian follicles deformed Upper part of trachea withhaemorrhages Enlarged liver Enlarged liver with haemorrhagesInflammation of kidneys Airsacculitis Haemorrhages in abdominal fatEnlarged spleen with haemorrhages Haemorrhages in thoracic cavityHaemorrhages in muscle Haemorrhages in oviduct Haemorrhages in the heartand hydropericardium Haemorrhages of coronary fat Haemorrhages in thethoracic cavity

Three strains of a novel type of Gram-negative, facultative anaerobic,pleomorphic rod-shaped bacteria, were deposited at the American TypeCulture Collection (ATCC), 1081, University Boulevard, Manassas, Va.20110-2209, USA, under deposit number: ATCC No. PTA-3667 for BIV-4985;ATCC No. PTA-3668 for BIV-AVICOR, and ATCC No. PTA-3669 for BIV-07990.The date of deposit was Aug. 22, 2001.

The deposited bacteria were typed according to standard determinationmethods, using Bergey's Manual of Systematic Bacteriology Volume I(1984. Williams and Wilkins, 428 East Preston Street. Baltimore, USA.)

TABLE 1 BIV-07990; ATCC No. PTA-3669 Macroscopic morphology Coloniesgrown on Sheep Blood Agar for 24 hours, range from 1.0 to 1.5 mm indiameter, bright translucent, low convex, smooth and creamy, βhemolysis. Microscopic morphology Gram-negative, non-motile, pleomorphicrods, often exhibit bipolar staining. Biochemical and other tests TESTREACTION Section A Oxidase + Catalase + Indole − Glucose + Sucrose +MacConkey + Urease − Nitrate + Section B Maltose + Mannitol + Arabinose− Celobiose − Sorbitol + Xilose + Treahalose − Salicin − Ornithine −Esculine − β Glucosidase − α-Fucosidase − β Galactosidase +

TABLE 2 BIV-AVICOR; ATCC No. PTA-3668 Macroscopic morphology Coloniesgrown on Sheep Blood Agar for 24 hours, range from 1.0 to 1.5 mm indiameter, bright translucent, low convex, smooth and creamy, βhemolysis. Microscopic morphology Gram-negative, non-motile, pleomorphicrods, often exhibit bipolar staining. Biochemical and other tests TESTREACTION Section A Oxidase + Catalase + Indole − Glucose + Sucrose +MacConkey + Urease − Nitrate + Section B Maltose + Mannitol + Arabinose− Celobiose − Sorbitol + Xylose + Trehalose + Salicin − Ornithine −Esculin − β Glucosidase +^(α) α-Fucosidase − β Galactosidase + ^(α)80%are positive

TABLE 3 BIV-4895; ATCC No. PTA-3667 Macroscopic morphology Coloniesgrown on Sheep Blood Agar for 24 hours, range from 1.0 to 1.5 mm indiameter, bright translucent, low convex, particulate and dry, βhemolysis. Microscopic morphology Gram-negative, non-motile, pleomorphicrods, often exhibit bipolar staining. Biochemical and other tests TESTREACTION Section A Oxidase + Catalase + Indole − Glucose + Sucrose +MacConkey + Urease − Nitrate + Section B Maltose − Mannitol + Arabinose− Celobiose − Sorbitol + Xylose + Trehalose + Salicin − Ornithine −Esculin − β Glucosidase − α-Fucosidase − β Galactosidase +

Identification of the Causative Agent

Bacteria were isolated from infected trachea, palatine cleft, ovary,liver, heart, kidney and gonads (broilers). They are identified asGallibacterium.

Beta haemolysis + Gram stain − Oxidase + Catalase + Mac Conkey + Urease− Nitrate + Indole −

Initial Biotyping:

Bacterial isolates were initially purified and biotyped according to themethod described by Jaworski et al. (1). Three different biotypes (4,2, 1) were identified.

Briefly, from the purified isolates, a single colony was inoculated intotubes containing 3 ml of Tryptose Broth and incubated at 37° C. for 8hours. A loop of inoculum (20 μl) from the tube was then transferredinto another tube containing 3 ml of 1% sugar to be tested and incubatedfor 7 days at 37° C. before results were recorded

Initial Challenge Model:

Following purification of the bacteria, isolates were grown in tryptosemedia to obtain large quantities of pure pathogens. In order to validateKoch postulates, 3 different groups (20 birds per group) of specificpathogen free (SPF) chicken 13 weeks of age were infected with eachbiotype (0.2 ml/bird; 3×10⁸ CFU/ml) by intravenous route. The birds wereobserved daily for 3 days for morbidity and mortality. At the end of the3^(rd) day, all birds were sacrificed, post-mortem lesions recorded andorgan samples (liver and gonads) were collected for re-isolation.Post-mortem lesions of birds that died were also recorded.

Biotyping According to Christensen et al.

In light of the on-going terminology changes in the art, the strains ofthe present application were subjected to phenotypical characterizationaccording to Christensen et al. (2003), including gram, urase enzymeactivity, motility, cytochrome oxidase activity, and haemolysis.Furthermore, all the strains were hybridized both withGallibacterium-specific probe GAN850, and its inverse and complementaryprobe, and the Gallibacterium-specific probe EUB338 according to Bojesenet al., Detection of Gallibacterium spp. In Chickens by Fluorescent 16SrRNA In Situ Hybridization, J. Clin. Microbiol., Vol. 41, No. 11,November 2003, p. 5167-5172. The results from both phenotypic andgenotypic characterization indicated that all the strains belonged togenus Gallibacterium.

Results

Clinical signs: Prostration, Lameness, Displaced comb, Ruffled feathers,Cyanosis at the tip of the comb.

Lesions:

BIV-4895 BIV-Avicor BIV-07990 Lesion (Biotype 4) (Biotype 2) (Biotype 1)Heart edema 73% 37% 90% Heart haemorrhages 90% — 70% Haemorrhages incoronary fat 90% 37% 50% Pericarditis 73% 46% 30% Haemorrhages inthoracic — 19% 40% cavity Haemorrhages in ovary 64%  9% 20% Inflammationof kidneys 64% 46% 60% Haemorrhages in kidneys 55% 46% 20% Enlargedliver with — 73% — haemorrhages Airsacculitis 64% — — Haemorrhages inmuscle — 37% — Mortality 46% 19% —

EXAMPLE II Growth of the Bacteria According to the Invention,Preparation of the Vaccine and Vaccination of SPF Birds

Strains were grown on Tryptose Broth (TB). Harvest was done atlogarithmic growth phase around 6-8 hours post-inoculation depending onthe strain. Plate count was made in sheep blood agar for titration.Colony forming units per mililiter (CFU/ml) was determined using 1:10dilutions of the harvest. Cells were killed by adding formaldehyde to afinal concentration of 0.2%. Following a sterility check of thissuspension, a minimal titer of 10⁸ CFU/ml was added to the finalvaccine.

The vaccine was prepared by mixing the two strains (BIV-4895, ATCC No.PTA-3667 and BIV-AVICOR, ATCC No. PTA-3668) and oil-adjuvant (awater-in-oil emulsion on the basis of a mineral oil with a ratio of 60%oil/40% water) to a minimal concentration of 10^(7.0) CFU/strain/ml.

Specific pathogen free (SPF) chicken were vaccinated at 2, 5 and 9 weeksof age by injection of 0.5 ml of the vaccine subcutaneously halfway downthe neck.

EXAMPLE III Preparation of Challenge Strains and Challenge of Vaccinatedand Control Groups

Bacterial strains BIV-4895, ATCC No. PTA-3667 and BIV-AVICOR, ATCC No.PTA-3668, were grown on sheep blood agar for 24 hrs. at 37° C. The cellswere harvested in Tryptose Broth (TB) until a suspension with an OpticalDensity of 2.0 was obtained, using a spectrophotometer at wavelength of540 nm. For challenge, preparations were made that contain the followingnumber of cells in the final challenge-volume:

3×10⁹ CFU/ml BIV-AVICOR; ATCC No. PTA-36681.45×10¹⁰ CFU/ml BIV-4895; ATCC No. PTA-3667

At 13 weeks of age, 20 vaccinated and 20 non-vaccinated birds werechallenged by intravenous route of 0.2 ml of the inoculum (at least10^(8.0) CFU/bird). Birds were observed for 3 days for morbidity andmortality. After three days of observation all the remaining birds weresacrificed and re-isolation of the bacteria from liver and gonads weremade from each bird. Post-mortem lesions of birds that died were alsorecorded.

Results

Mortality + Group of birds Challenge inoculum Reisolation Protection %Control Negative N/A 0 N/A Control Positive ATCC No. PTA-3667 77 23Control Positive ATCC No. PTA-3668 54 46 Vaccinated ATCC No. PTA-3667 0100 Vaccinated ATCC No. PTA-3668 5 95

EXAMPLE IV Growth of the Bacteria According to the Invention,Preparation Of the Vaccine and Vaccination of SPF Birds

Strains were grown on Tryptose Broth (TB). Harvest was done atlogarithmic growth phase around 6-8 hours post-inoculation depending onthe strain. Plate count was made in sheep blood agar for titration.Colony forming units per mililiter (CFU/ml) was determined using 1:10dilutions of the harvest. Cells were killed by adding formaldehyde to afinal concentration of 0.2%. Following a sterility check of thissuspension, a minimal titer of 10⁸ CFU/ml was added to the finalvaccine.

The vaccine was prepared by mixing the three strains (BIV-4895, ATCC No.PTA-3667; BIV-AVICOR, ATCC No. PTA-3668 and BIV-07990, ATCC No.PTA-3669) and oil-adjuvant (a water-in-oil emulsion on the basis of amineral oil with a ratio of 60% oil/40% water) to a minimalconcentration of 10^(7.0) CFU/strain/ml.

Specific pathogen free (SPF) chicken were vaccinated at 2, 5 and 9 weeksof age by injection of 0.5 ml of the vaccine subcutaneously halfway downthe neck.

EXAMPLE V Preparation of Challenge Strains and Challenge of Vaccinatedand Control Groups

Bacterial strains BIV-4895, ATCC No. PTA-3667; BIV-AVICOR, ATCC No.PTA-3668 and BIV-07990, ATCC No. PTA-3669, were grown on sheep bloodagar for 24 hrs. at 37° C. The cells were harvested in Tryptose Broth(TB) until a suspension with an Optical Density of 2.0 was obtained,using a spectrophotometer at wavelength of 540 nm. For challenge,preparations were made that contain the following number of cells in thefinal challenge-volume:

8.3×10⁹ CFU/ml BIV-AVICOR; ATCC No. PTA-3668

2.2×10⁹ CFU/ml BIV-4895; ATCC No. PTA-3667

1.0×10¹⁰ CFU/ml BIV-07990; ATCC No. PTA-3669

At 13 weeks of age, 20 vaccinated and 20 non-vaccinated birds werechallenged by intravenous route of 0.2 ml of the inoculum (at least10^(8.0) CFU/bird). Birds were observed for 3 days for morbidity andmortality. After three days of observation all the remaining birds weresacrificed and re-isolation of the bacteria from liver and gonads weremade from each bird. Post-mortem lesions of birds that died were alsorecorded.

Results

Mortality + Group of Birds Challenge Inoculum Reisolation Protection (%)Control Negative N/A 0 N/A Vaccinated Control Negative N/A 0 N/ANon-vaccinated Vaccinated ATCC No. PTA-3669 27.3 72.7 Vaccinated ATCCNo. PTA-3668 20.9 79.1 Vaccinated ATCC No. PTA-3667 16.7 83.7 ControlPositive ATCC No. PTA-3669 53.3 46.7 Control Positive ATCC No. PTA-366853.3 46.7 Control Positive ATCC No. PTA-3667 64.3 35.7

Serological Test

Hyperimmune sera were produced in rabbits with isolate representing eachbiotype, according to the method of Biberstein et. al. (Biberstein E L.,Meyer M. E., and Kenedy P. C. Colonial variation of Pasteurellahaemolytica isolated from sheep. J. Bact. 1958, 76: 445-452.)

The isolates were grown on blood agar overnight, then harvested insaline containing 0.3% formalin. The cells were washed once and adjustedto 10% transmittance at 575 nm for injection. The injections were by IVroute according to the following schedule: 0.5 ml, 1.0, 2.0, 3.0, 3.0,3.0 at 4 day intervals and all rabbits were bled 4 days after the finalinjection.

The hyperimmune serum was tested for their specificity using the 3biotype strains and were reacted with homologous and heterologous rabbitantiserum (2 fold dilutions) by rapid plate agglutination.

Antiserum of each biotype was diluted until the end point was reached todetermine the highest dilution that was positive.

Dilution (log²) Antigen Biotype 1 Antiserum 1 2 3 4 5 6 7 8 9 10 111 + + + + + + + + + + + 2 − − − − − − − − − − − 4 − − − − − − − − − − −

Dilution (log²) Antigen Biotype 4 Antiserum 1 2 3 4 5 6 7 8 9 10 11 1 −− − − − − − − − − − 2 − − − − − − − − − − − 4 + + + + + + + + + + +

Dilution (log²) Antigen Biotype 2 Antiserum 1 2 3 4 5 6 7 8 9 10 11 1 −− − − − − − − − − − 2 + + + + + + + + + + + 4 − − − − − − − − − − −

The biotype specific hyperimmune sera was then used as positive controlin micro-plate serum agglutination test.

EXAMPLE VI Preparation of Challenge Strains and Challenge of Vaccinatedand Control Groups

Bacterial strains BIV-4895, ATCC No. PTA-3667, BIV-AVICOR, ATCC No.PTA-3668 and BIV-07990, ATCC No. PTA-3669, were grown on sheep bloodagar for 24 hrs. at 37° C. The cells were harvested in Tryptose Broth(TB) until a suspension with an Optical Density of 2.0 was obtained,using a spectrophotometer at wavelength of 540 nm. For challenge,preparations were made that contain the following number of cells in thefinal challenge-volume:

1.5×10¹⁰ CFU/ml BIV-AVICOR; ATCC No. PTA-3668

1.7×10¹⁰ CFU/ml BIV-4895; ATCC No. PTA-3667

1.6×10¹⁰ CFU/ml BIV-07990; ATCC No. PTA-3669

At 13 weeks of age, 20 vaccinated and 20 non-vaccinated birds werechallenged by intravenous route of 0.2 ml of the inoculum (at least10^(8.0) CFU/bird). Birds were observed for 3 days for morbidity andmortality. After three days of observation all the remaining birds weresacrificed, post mortem lesions were recorded and re-isolation of thebacteria from liver, heart and gonads were made from each bird.

Results

TABLE 1 Evaluation on the effect of vaccine based on mortality andre-isolation. Challenge Mortality + Group of birds inoculum ReisolationProtection % Control negative non- N/A N/A N/A vaccinated ControlPositive BIV-4895 70 30 Control Positive BIV-AVICOR 80 20 ControlPositive BIV-07990 88.8 11.2 Vaccinated BIV-4895 10 90 VaccinatedBIV-AVICOR 10 90 Vaccinated BIV-07990 15 85

TABLE 2 Evaluation on the effect of vaccine based on gross lesionsfollowing challenge. Challenge Group of birds inoculum % of LesionsProtection % Control negative non- N/A N/A N/A vaccinated ControlPositive BIV-4895 74.4 25.6 Control Positive BIV-AVICOR 27.0 73.0Control Positive BIV-07990 7.0 93.0 Vaccinated BIV-4895 4.0 96.0Vaccinated BIV-AVICOR 1.1 99.0 Vaccinated BIV-07990 2.4 98.0

1. Gram-negative, facultative anaerobic, pleomorphic rod-shaped bacteriacausing a disease of the upper respiratory and of the reproductive tractof poultry, wherein the bacteria are selected from the group ofPasteurella trehalosi and Mannheimia haemolytica.
 2. Pasteurellatrehalosi or Mannheimia haemolytica, wherein the Pasteurella orMannheimia are beta haemolysis-positive, Gram-negative,oxidase-positive, catalase-positive, urease-negative, nitrate-positiveand indole-negative.
 3. Pasteurella trehalosi or Mannheimia haemolyticaaccording to claim 2, wherein the Pasteurella or Mannheimia areMacConkey positive.
 4. Pasteurella trehalosi or Mannheimia haemolyticaaccording to claim 2, wherein the Pasteurella or Mannheimia areglucose-positive, sucrose-positive, mannitol-positive,arabinose-negative, celobiose-negative, xylose-positive,salicin-negative, ornithine-negative, esculin-negative,alpha-fucosidase-negative, and beta-galactosidase-positive. 5.Pasteurella trehalosi according to claim 2, wherein the Pasteurella isArabinose-negative and Trehalose-positive.
 6. Mannheimia haemolyticaaccording to claim 2, wherein the Mannheimia is Arabinose-negative andTrehalose-negative.
 7. A method for inactivation of a Pasteurellatrehalosi or Mannheimia haemolytica according to claim 1 comprising theuse of formaldehyde to a final concentration of 0.2%.
 8. InactivatedPasteurella trehalosi or Mannheimia haemolytica as recited in claim 1.9. Inactivated Pasteurella trehalosi or Mannheimia haemolytica accordingto claim 8, wherein the Pasteurella trehalosi or Mannheimia haemolyticaare selected from the group of Pasteurella trehalosi ATCC No. PTA-3667,Pasteurella trehalosi ATCC No. PTA-3668 and Mannheimia haemolytica ATCCNo. PTA-3669.
 10. Live, attenuated Pasteurella trehalosi or Mannheimiahaemolytica as recited in claim
 1. 11. Live, attenuated Pasteurellatrehalosi or Mannheimia haemolytica according to claim 10, wherein thePasteurella trehalosi or Mannheimia haemolytica are selected from thegroup of Pasteurella trehalosi ATCC No. PTA-3667, Pasteurella trehalosiATCC No. PTA-3668 and Mannheimia haemolytica ATCC No. PTA-3669. 12.Fragments or fractions containing at least one antigen of Pasteurellatrehalosi or Mannheimia haemolytica according to claim
 1. 13. Fragmentsor fractions according to claim 12, wherein the fragments contain atleast one antigen from bacteria selected from the group of Pasteurellatrehalosi ATCC No. PTA-3667, Pasteurella trehalosi ATCC No. PTA-3668 andMannheimia haemolytica ATCC No. PTA-3669.
 14. Vaccine compositioncomprising an inactivated Pasteurella trehalosi or Mannheimiahaemolytica according to claim
 9. 15. Vaccine composition comprisingfragments or fractions of said Pasteurella trehalosi or Mannheimiahaemolytica according to claim
 12. 16. Vaccine composition comprisinglive, attenuated Pasteurella trehalosi or Mannheimia haemolyticaaccording to claim
 10. 17. Vaccine composition according to claim 14,further comprising one or more suitable adjuvant(s) or excipient(s) orcarrier(s).
 18. Vaccine composition according to claim 15, furthercomprising one or more suitable adjuvant(s) or excipient(s) orcarrier(s).
 19. Vaccine composition according to claim 18, furthercomprising at least one other antigen from a virus or microorganismpathogenic to poultry.
 20. Vaccine composition according to claim 16,further comprising one or more suitable adjuvant(s) or excipient(s) orcarrier(s).
 21. Method of diagnosis of a disease comprising the steps ofobtaining a sample from poultry affected by the disease, wherein thesample is selected from the group of blood, serum, plasma, tissuescrapings, washings, swabs, and tissue, and analysing the sample for thepresence of Pasteurella trehalosi or Mannheimia haemolytica as recitedin claim 1.